A comparison of the Sepsis-2 and Sepsis-3 definitions for assessment of mortality risk in dogs with parvovirus.

OBJECTIVE: To evaluate the use of a modified Sepsis-3 (mSepsis-3) definition compared to the currently used modified Sepsis-2 (mSepsis-2) definition to determine whether the mSepsis-2 or mSepsis-3 stratifications were able to identify populations of dogs ultimately more likely to die from canine parvovirus (CPV) infection. DESIGN: Retrospective, January 2009 to March 2020. SETTING: A private, small animal, urban, referral emergency and specialty hospital. ANIMALS: Fifty-nine client-owned dogs hospitalized for treatment of CPV. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Dogs were divided into mSepsis-2 and mSepsis-3 categories based on the highest level of illness severity reached during hospitalization. Greater illness severity based on mSepsis-2 criteria (ie, sepsis, severe sepsis, septic shock) was associated with an increase in average length of stay (P < 0.001), increase in average cost of stay (P < 0.01), and presence of leukopenia (P < 0.05). An increase in illness severity within the mSepsis-2 criteria was not associated with hyperlactatemia (P = 0.29), presence of neutropenia (P = 0.12), or mortality (P = 0.35). Greater illness severity based on mSepsis-3 criteria (ie, infection only, sepsis, septic shock) was associated with an increase in mortality (P < 0.05), increase in average length of stay (P < 0.001), increase in average cost of stay (P < 0.01), presence of leukopenia (P < 0.01), and presence of neutropenia (P < 0.05). The mSepsis-3 criteria were not associated with the presence of hyperlactatemia (P = 0.68). There was no significant difference between survivors and nonsurvivors in the presence of leukopenia (P = 0.19), neutropenia (P = 0.67), or hyperlactatemia (P = 0.58). CONCLUSIONS: The mSepsis-3 diagnostic criteria appear to better identify dogs with CPV at higher risk for mortality compared to the mSepsis-2 criteria.

Why serology just is not enough: Strategic parvovirus risk assessment using a novel qPCR assay.

Health monitoring of laboratory rodents not only improves animal health but also enhances the validity of animal experiments. In particular, infections of laboratory animals with murine parvoviruses influence biomedical research data. Despite strict barrier housing, prevalence remains high in animal facilities, leading to increased risk of parvovirus introduction after the import of contaminated mice. Unfortunately, hygienic rederivation can be challenging, since gametes often contain residual virus material. Consequently, the process has to be closely monitored with highly sensitive diagnostic methods to verify parvovirus decontamination of the rederived progeny. However, diagnostic sensitivity of traditional methods is often low and requires testing of large animal cohorts. Therefore, we aimed to develop a powerful quantitative real-time polymerase chain reaction (qPCR) assay for the fast and reliable detection of murine parvoviruses in different sample materials. We validated the assay within an infection experiment and systematically analysed various animal-derived and environmental sample materials. We further developed a strategic risk assessment procedure for parvovirus monitoring after embryo transfer. Our novel qPCR assay reliably detected parvovirus DNA in a broad variety of sample materials, with environmental samples dominating in the acute phase of infection, whereas animal-derived samples were more suitable to detect low virus loads in the chronic phase. Here, the assay served as a highly sensitive screening method for parvovirus contamination in mouse colonies, requiring significantly lower sample sizes than traditional methods like conventional PCR and serology. Thus, the use of our novel qPCR assay substantially improves parvovirus diagnostics, enhancing research validity according to the 6Rs.

Assessment of certain biomarkers for predicting survival in response to treatment in dogs naturally infected with canine parvovirus.

Canine parvovirus (CPV) enteritis is an important cause of morbidity and mortality in puppies despite aggressive treatment. Identification of reliable biomarkers for CPV enteritis is essential to determine the severity, duration of hospitalization, and predict the clinical outcome. Meanwhile, the biomarkers will assist in decision-making with clients about the further course of treatment or euthanasia. The present study was conducted to evaluate the changes of total leukocyte count (TLC), neutrophil count, and serum concentrations of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), intestinal fatty acid binding protein-2 (IFABP-2), albumin, ceruloplasmin (Cp), cortisol, free triiodothyronine (FT3) and free thyroxine (FT4) in survivors and non-survivors as a predictor of the clinical outcome. Marked leukopenia, neutropenia, hypoalbuminemia, elevated levels of CK-MB, IFABP-2, Cp, and cortisol were noticed in CPV-infected dogs than healthy dogs but, LDH, FT3 and FT4 concentrations did not differ significantly. The CPV-infected non-survivors had persistent leukopenia, neutropenia and elevated CK-MB, IFABP-2, Cp and cortisol concentrations at 72 h of commencement of treatment. In CPV-infected survivors, TLC and neutrophil count were significantly increased, and CK-MB, IFABP-2, Cp and cortisol concentrations were significantly decreased at 72 h of commencement of treatment. The positive predictive values (PPVs) for survival using cut-off value of TLC (>3.2 × 103/µL), neutrophil count (>1.65 × 103/µL), CK-MB (≤234.50 U/L), IFABP-2 (≤7.61 ng/mL), Cp (≤0.605 g/L) and cortisol (≤16.90 ng/mL) were determined as 89.47%, 88.88%, 94.73%, 93.33%, 94.44% and 89.47%, respectively with better area under receiver operating characteristic (ROC) curve as well as sensitivity. The magnitude of decrease in TLC, neutrophil count, and increase in CK-MB, IFABP-2, Cp and cortisol concentrations at 72 h of initiation of treatment in dogs with parvoviral enteritis could be useful indicators for the prognosis of the disease. Based on sensitivity (%) and specificity (%) from ROC curve analysis and PPV (%), it is concluded that serum CK-MB concentration will serve as the most useful biomarker followed by Cp and absolute neutrophil count.

Simultaneous detection of 4 prototypic rat parvoviruses using the luminex xTAG assay in laboratory animal health monitoring.

There are currently four rat parvoviruses including Kilham rat virus (KRV), Toolans H-1 parvovirus (H-1virus), rat parvovirus type 1a (RPV-1a) and rat minute virus (RMV). Virus detection methods are commonly based on conventional PCR - agarose gel electrophoresis or serological assay methods These methods are both time-consuming and lack specificity. In this study, we developed a bead array xTAG assay for the simultaneous detection and discrimination of four rat parvoviruses. The detection limits ranged from 100 to 1000 copies/µL of input purified plasmid DNA. We examined 50 clinical specimens and 15 facal samples by xTAG assay and conventional PCR. The results showed a high consistency except for several weak positive infections. It demonstrated that the xTAG-multiplex PCR method is specific, sensitive and suitable for high throughput platforms for rat parvovirus screening of clinical samples and contaminated biological materials.

Achieving a Successful Scale-Down Model and Optimized Economics through Parvovirus Filter Validation using Purified TrueSpikeTM Viruses.

UNLABELLED: This article describes a four virus panel validation of EMD Millipore's (Bedford, MA) small virus-retentive filter, Viresolve® Pro, using TrueSpike(TM) viruses for a Biogen Idec process intermediate. The study was performed at Charles River Labs in King of Prussia, PA. Greater than 900 L/m(2) filter throughput was achieved with the approximately 8 g/L monoclonal antibody feed. No viruses were detected in any filtrate samples. All virus log reduction values were between ≥3.66 and ≥5.60. The use of TrueSpike(TM) at Charles River Labs allowed Biogen Idec to achieve a more representative scaled-down model and potentially reduce the cost of its virus filtration step and the overall cost of goods. The body of data presented here is an example of the benefits of following the guidance from the PDA Technical Report 47, The Preparation of Virus Spikes Used for Viral Clearance Studies. LAY ABSTRACT: The safety of biopharmaceuticals is assured through the use of multiple steps in the purification process that are capable of virus clearance, including filtration with virus-retentive filters. The amount of virus present at the downstream stages in the process is expected to be and is typically low. The viral clearance capability of the filtration step is assessed in a validation study. The study utilizes a small version of the larger manufacturing size filter, and a large, known amount of virus is added to the feed prior to filtration. Viral assay before and after filtration allows the virus log reduction value to be quantified. The representativeness of the small-scale model is supported by comparing large-scale filter performance to small-scale filter performance. The large-scale and small-scale filtration runs are performed using the same operating conditions. If the filter performance at both scales is comparable, it supports the applicability of the virus log reduction value obtained with the small-scale filter to the large-scale manufacturing process. However, the virus preparation used to spike the feed material often contains impurities that contribute adversely to virus filter performance in the small-scale model. The added impurities from the virus spike, which are not present at manufacturing scale, compromise the scale-down model and put into question the direct applicability of the virus clearance results. Another consequence of decreased filter performance due to virus spike impurities is the unnecessary over-sizing of the manufacturing system to match the low filter capacity observed in the scale-down model. This article describes how improvements in mammalian virus spike purity ensure the validity of the log reduction value obtained with the scale-down model and support economically optimized filter usage.

RUNspike, a complementary virus filter spiking method: a solution to the problem of reduced throughput due to the addition of the virus spike.

The traditional approach to virus filter spiking studies (virus added to the feed solution before the start of filtration) can lead to oversized viral filtration systems because of the non-representative volumetric throughputs (L/m2) that can be seen with the addition of the virus spike. The reduction in throughput is thought to be caused by interactions that take place between the species in the virus stock solution alone, or in conjunction with the species in the drug product. The traditional approach assumes that virus filter log reduction value (LRV) is directly related to volumetric throughput and limits manufacturing scale designs to the volumetric throughput achieved in the spiking study. This article references previous work that shows that % flow decay is a more relevant critical parameter than volumetric throughput for a poly(vinylidene diflouride) (PVDF) parvovirus filter. Based on this work, one could design the manufacturing-scale viral filtration system to the representative volumetric throughput achieved without the virus spike and implement a flow decay limit in manufacturing to ensure virus LRV. RUNspike is a complementary method that can be easily implemented today and that goes one step further. The RUNspike method challenges the same total virus as the traditional method, but the virus is added at the end of the filtration, after the representative volumetric throughput has been demonstrated. Comparable RUNspike and traditional LRV data, at the same flow decay, bridges the gap and strengthens the case for the flow decay-based design approach.

The need for continuing vigilance: addressing the threat for transmission of blood-borne infectious disease.

As international travel and human encroachment into previously isolated areas have increased, so too has the potential for the emergence of new infectious diseases. Populations likely to be susceptible to new infectious diseases have also increased in size. The past three decades have seen outbreaks of diseases caused by parvoviruses, Nipah virus, circoviruses, and prions. Infectious pathogens such as these are formidable opponents; they can adapt to new hosts or cause variant diseases within new hosts. Many are also resistant to current inactivation techniques. In order to prevent or contain outbreaks, pathogens that emerge must be identified quickly and efficiently; research and ongoing global surveillance are therefore of primary importance. To effectively protect the blood supply and blood-based therapies, this research should include investigations into improved techniques for detection, screening, and viral inactivation, as well as into ways to reduce patient exposure to infectious pathogens via therapeutic agents. The proactive devotion of appropriate resources to infectious disease containment and prevention prior to an epidemic should be perceived as both essential public health policy and cost effective.

Rodent vendor apparent source of mouse parvovirus in sentinel mice.

Mouse parvovirus (MPV) has been increasingly prevalent in laboratory animal facilities, and the source of infection often can be difficult to determine. After 4 years of sporadic MPV detected in our sentinel mice and continual failure to identify index cases in colony mice, we developed a regimen to house newly arrived vendor mice in large sterile cages with a high stocking density. Some of these mice were retained in isolation after the remaining mice were deployed as sentinels. After detecting MPV seropositive sentinel mice 4 weeks after introduction to the mouse colonies in one facility, the remaining naïve mice that had been previously housed with those sentinels also tested positive for MPV, despite never having been exposed to colony mice. These results suggest that commercially bred mice intended for use as sentinels may, in fact, arrive at animal facilities already infected with MPV. Depending upon numerous factors, including the health surveillance methods used, it is possible that a low prevalence of MPV may exist undetected at rodent vendors.

Risk assessment of the use of autonomous parvovirus-based vectors.

Autonomous parvoviruses are small, non-enveloped, lytic DNA viruses replicating in the nucleus of actively dividing mammalian cells of appropriate species and tissue origins. In contrast to AAV, the other main subgroup of parvoviruses, autonomous parvoviruses do not require the assistance of an auxiliary virus for productive infection and do not stably integrate in the cellular DNA. Therefore, autonomous parvoviruses are suitable vectors for mediating transient gene transduction in dividing target cells. Interestingly, some of these viruses possess a striking inherent oncotropism, which may render them particularly suitable as selective vehicles in the clinical context of cancer gene therapy. In this chapter, we will present a brief overview of the biology of autonomous parvoviruses. This topic will be followed by a description of the design and recent developments in the production and use of parvoviral vectors, with a particular emphasis on biosafety aspects. Finally, the risk assessment related to the production and use of parvoviral vectors will be discussed in last part of the chapter.

Inactivation of parvovirus B19 in coagulation factor concentrates by UVC radiation: assessment by an in vitro infectivity assay using CFU-E derived from peripheral blood CD34+ cells.

BACKGROUND: Nonenveloped and thermostable viruses such as parvovirus B19 (B19) can be transmitted to patients who are receiving plasma-derived coagulation factor concentrates treated by the S/D method for inactivating enveloped viruses. Therefore, it is important to develop and validate new methods for the inactivation of nonenveloped viruses. STUDY DESIGN AND METHODS: Suspensions of B19 in coagulation factor concentrates (FVIII) were irradiated with UVC light. B19 infectivity was determined by an indirect immunofluorescence assay using CFU-E, as a host cell, derived from peripheral blood CD34+ cells. The effects of catechins on B19 infectivity and on FVIII activity after UVC illumination were also examined. RESULTS: The indirect immunofluorescence assay estimated the B19 infectivity of samples containing virus copies of 10(5) to 10(11) per 10 microL to be a median tissue culture-infectious dose of 10(0.3) to 10(5.4) per 10 microL. B19 was inactivated by 3 log at 750 J per m(2) of UVC radiation and was undetectable after 1000 or 2000 J per m(2) of irradiation. However, FVIII activity decreased to 55 to 60 percent of pretreatment activity after 2000 J per m(2) of UVC radiation. This was inhibited in the presence of rutin or catechins. Epigallocatechin gallate could maintain FVIII activity at almost 100 percent of pretreatment activity after 2000 J per m(2) of UVC radiation, while B19 infectivity was decreased to undetectable levels, which resulted in >3.9 log inactivation. CONCLUSION: UVC radiation in the presence of catechins, especially epigallocatechin gallate, appears to be an effective method of increasing the viral safety of FVIII concentrates without the loss of coagulation activity.